Methods of Preparing Dual-Indexed DNA Libraries for Bisulfite Conversion Sequencing

ABSTRACT

Described herein are methods of preparing dual-indexed nucleic acid libraries for methylation profiling using bisulfite conversion sequencing. In various embodiments, the methods use a two-step indexing process to tag bisulfite-treated DNA with unique molecular identifiers (UMIs).

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims benefit of priority of U.S. ProvisionalApplication Ser. No. 62/373,261 filed on Aug. 10, 2016, and U.S.Provisional Application Ser. No. 62/397,650 filed on Sep. 21, 2016, bothof which are herein incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

DNA methylation plays an important role in regulating gene expression.Aberrant DNA methylation has been implicated in many disease processes,including cancer. DNA methylation profiling using bisulfite conversionsequencing is increasingly recognized as a valuable diagnostic tool fordetection and diagnosis of cancer. For example, specific patterns ofdifferentially methylated regions and/or allele specific methylationscan be used as molecular markers for non-invasive diagnostics usingcirculating cell-free DNA. However, amplification and/or sequencingartifacts (errors) introduced during library preparation and/orsequencing can bias the results of DNA methylation analysis. There is aneed for new methods of preparing a DNA library for bisulfite conversionsequencing for methylation profiling.

SUMMARY OF THE INVENTION

Disclosed herein are methods of preparing dual-indexed nucleic acidlibraries for methylation profiling using bisulfite or enzymaticconversion sequencing. In various embodiments, the methods use atwo-step indexing process to tag bisulfite-treated or enzymaticallyconverted DNA (in which unmethylated cytosines are converted to uracilsin the converted DNA) with unique molecular identifiers (UMIs), whereina first UMI is added to converted DNA using a single-strand DNA (ssDNA)ligation reaction and a second UMI is added in a subsequent processingstep (e.g., a double-strand ligation step). The UMIs are used toidentify individual DNA molecules and reduce or substantially eliminatesequencing and/or amplification-induced artifacts (based on a consensusamong reads sharing the same UMI) thereby increasing the accuracy of DNAmethylation analysis.

In certain embodiments, described herein, is a method of determining amethylation profile of an individual comprising: obtaining a biologicalsample from the individual; converting unmethylated cytosines to uracilsin nucleic acid molecules of the biological sample to produce bisulfiteconverted nucleic acid molecules; determining the nucleic acid sequenceof the converted nucleic acid molecules; and comparing the nucleic acidsequence of the converted nucleic acid molecules to a reference nucleicacid sequence, to determine the methylation profile of the individual.In certain embodiments, the individual is a human. In certainembodiments, the biological sample comprises blood, serum, plasma,urine, cerebral spinal fluid, or lymph. In certain embodiments,converting unmethylated cytosines to uracils comprises incubation withbisulfite ion. In certain embodiments, the nucleic acid molecules areDNA. In certain embodiments, the DNA is cell-free DNA. In certainembodiments, the cell-free DNA is derived from a linear chromosome. Incertain embodiments, the linear chromosome is an autosome. In certainembodiments, the method further comprises ligating a nucleic acidadapter to the converted nucleic acids. In certain embodiments, thenucleic acid adapter comprises a unique molecular identifier. In certainembodiments, the nucleic acid adapter comprises a universal primingsite. In certain embodiments, the nucleic acid adapter is singlestranded. In certain embodiments, the nucleic acid adapter is partiallysingle stranded and partially double stranded. In certain embodiments,ligating the nucleic acid adapter is performed on single strandednucleic acids. In certain embodiments, the nucleic acid adapter isattached to a solid support. In certain embodiments, the solid supportis a bead. In certain embodiments, the bead is heat labile. In certainembodiments, the method further comprises performing primer extension ofthe converted nucleic acid molecules, unconverted nucleic acidmolecules, or both. In certain embodiments, the method further comprisesligating a nucleic acid adapter to a second end of the converted nucleicacid molecules. In certain embodiments, ligating the nucleic acidadapter to a second end of the converted nucleic acid molecules createsa gap between a 5′ end of the converted nucleic acid molecules and a 3′end of the third adapter most proximal to the 5′ end of the convertednucleic acid molecules. In certain embodiments, the method furthercomprises amplifying the converted nucleic acid molecules beforedetermining the nucleic acid sequence. In certain embodiments, theamplifying comprises polymerase chain reaction. In certain embodiments,the amplifying results in the addition of sequencing adapters to theconverted nucleic acid molecules, the unconverted nucleic acidmolecules, or both. In certain embodiments, determining the nucleic acidsequence comprises next-generation sequencing. In certain embodiments,determining the nucleic acid sequence comprises sequencing by synthesis,pyrosequencing, or ion semi-conductor sequencing. In certainembodiments, determining the nucleic acid sequence comprises sequencingto a depth of at least 10,000×. In certain embodiments, the methylationprofile is used to screen for or diagnose an autoimmune disease. Incertain embodiments, the methylation profile is used to screen for ordiagnose cancer. In certain embodiments, the methylation profile is usedto screen for or diagnose organ damage, organ disease, or organ failure.In certain embodiments, the methylation profile is used to screen for ordiagnose transplant rejection. In certain embodiments, the methodfurther comprises using the methylation profile in combination with anyof family history, clinical data, genome sequencing data, proteomicdata, or microbiome data to screen for or diagnose autoimmune disease,cancer, organ failure, or organ transplant rejection.

In certain embodiments, described herein is a method of determining amethylation profile of an individual comprising: (a) obtaining abiological sample from the individual; (b) converting unmethylatedcytosines to uracils in nucleic acid molecules of the biological sampleto produce converted nucleic acid molecules; (c) ligating a nucleic acidadapter comprising a unique molecular identifier to the convertednucleic acids; (d) determining the nucleic acid sequence of theconverted nucleic acid molecules; and (e) comparing the nucleic acidsequence of the converted nucleic acid molecules to a reference nucleicacid sequence, to determine the methylation profile of the individual.In certain embodiments, the biological sample comprises blood, serum,plasma, urine, cerebral spinal fluid, or lymph. In certain embodiments,the method further comprises enriching the converted nucleic acidmolecules, wherein the enrichment increases the amount of more targetmolecules compared to non targeted. In certain embodiments, convertingunmethylated cytosines to uracils comprises incubation with bisulfiteion. In certain embodiments, converting unmethylated cytosines touracils comprises incubation with a cytidine deaminase. In certainembodiments, the nucleic acid molecules are DNA. In certain embodiments,the DNA is cell-free DNA (cfDNA). In certain embodiments, the nucleicacid adapter comprises a universal priming site. In certain embodiments,the nucleic acid adapter is partially single stranded and partiallydouble stranded. In certain embodiments, ligating the nucleic acidadapter is performed on single stranded nucleic acids. In certainembodiments, nucleic acid adapter is attached to a solid support. Incertain embodiments, the solid support is a bead. In certainembodiments, the method further comprises performing primer extension ofthe converted nucleic acid molecules, unconverted nucleic acidmolecules, or both. In certain embodiments, the method further comprisesligating a nucleic acid adapter to a second end of the converted nucleicacid molecules. In certain embodiments, ligating the nucleic acidadapter to a second end of the converted nucleic acid molecules createsa gap between a 5′ end of the converted nucleic acid molecules and a 3′end of the third adapter most proximal to the 5′ end of the convertednucleic acid molecules. In certain embodiments, the method furthercomprises amplifying the converted nucleic acid molecules beforedetermining the nucleic acid sequence. In certain embodiments,amplifying comprises polymerase chain reaction. In certain embodiments,the amplifying results in the addition of sequencing adapters to theconverted nucleic acid molecules, the unconverted nucleic acidmolecules, or both. In certain embodiments, determining the nucleic acidsequence comprises next-generation sequencing. In certain embodiments,determining the nucleic acid sequence comprises sequencing to a depth ofat least 10,000×. In certain embodiments, the methylation profile isused to screen for or diagnose cancer. In certain embodiments, themethylation profile is used determine a tissue or origin of a cell-freeDNA.

In certain embodiments, described herein, is a method of determining amethylation profile of an individual comprising: obtaining a biologicalsample from the individual that is divided into at least two aliquots;converting unmethylated cytosines to uracils in nucleic acid moleculesof a first aliquot to produce a first aliquot comprising bisulfiteconverted nucleic acid molecules; determining the nucleic acid sequenceof the converted nucleic acid molecules and the nucleic acid moleculesof a second aliquot comprising unconverted nucleic acid moleculeswherein the unmethylated cytosines are not converted to uracils; andcomparing the nucleic acid sequence of the converted nucleic acidmolecules and the unconverted nucleic acid molecules, to determine themethylation profile of the individual. In certain embodiments, theindividual is a human. In certain embodiments, the biological samplecomprises blood, serum, plasma, urine, cerebral spinal fluid, or lymph.In certain embodiments, converting unmethylated cytosines to uracilscomprises incubation with bisulfite ion. In certain embodiments, the atleast two aliquots are at least about equal. In certain embodiments, thenucleic acid molecules are DNA. In certain embodiments, the DNA iscell-free DNA. In certain embodiments, the cell-free DNA is derived froma linear chromosome. In certain embodiments, the linear chromosome is anautosome. In certain embodiments, the method further comprises ligatinga nucleic acid adapter to the converted nucleic acids, the unconvertednucleic acids, or both. In certain embodiments, the nucleic acid adaptercomprises a unique molecular identifier. In certain embodiments, thenucleic acid adapter comprises a universal priming site. In certainembodiments, the nucleic acid adapter is single stranded. In certainembodiments, the nucleic acid adapter is partially single stranded andpartially double stranded. In certain embodiments, ligating the nucleicacid adapter is performed on single stranded nucleic acids. In certainembodiments, the nucleic acid adapter is attached to a solid support. Incertain embodiments, the solid support is a bead. In certainembodiments, the bead is heat labile. In certain embodiments, the methodfurther comprises combining the at least two aliquots before determiningthe nucleic acid sequence. In certain embodiments, the method furthercomprises performing primer extension of the converted nucleic acidmolecules, unconverted nucleic acid molecules, or both. In certainembodiments, the method further comprises ligating a nucleic acidadapter to a second end of the converted nucleic acid molecules, theunconverted nucleic acid molecules, or both. In certain embodiments,ligating the nucleic acid adapter to a second end of the convertednucleic acid molecules, the unconverted nucleic acid molecules, or bothcreates a gap between a 5′ end of the converted nucleic acid molecules,the unconverted nucleic acid molecules, or both and a 3′ end of thethird adapter most proximal to the 5′ end of the converted nucleic acidmolecules, the unconverted nucleic acid molecules, or both. In certainembodiments, the method further comprises amplifying the convertednucleic acid molecules, the unconverted nucleic acid molecules, or bothbefore determining the nucleic acid sequence. In certain embodiments,the amplifying comprises polymerase chain reaction. In certainembodiments, the amplifying results in the addition of sequencingadapters to the converted nucleic acid molecules, the unconvertednucleic acid molecules, or both. In certain embodiments, determining thenucleic acid sequence comprises next-generation sequencing. In certainembodiments, determining the nucleic acid sequence comprises sequencingby synthesis, pyrosequencing, or ion semi-conductor sequencing. Incertain embodiments, determining the nucleic acid sequence comprisessequencing to a depth of at least 10,000×. In certain embodiments, thenucleic acid sequence of the converted nucleic acid molecules and theunconverted nucleic acid molecules are compared to determine cytosinesfrom the nucleic acid molecules of the first aliquot that were convertedto uracils based upon a sequence of the nucleic acid adapter. In certainembodiments, the methylation profile is used to screen for or diagnosean autoimmune disease. In certain embodiments, the methylation profileis used to screen for or diagnose cancer. In certain embodiments, themethylation profile is used to screen for or diagnose organ damage,organ disease, or organ failure. In certain embodiments, the methylationprofile is used to screen for or diagnose transplant rejection. Incertain embodiments, the method further comprises using the methylationprofile in combination with any of family history, clinical data, genomesequencing data, proteomic data, or microbiome data to screen for ordiagnose autoimmune disease, cancer, organ failure, or organ transplantrejection.

In certain embodiments, described herein, is a method of preparing anucleic acid library for bisulfite conversion sequencing comprising:converting unmethylated cytosines to uracils in nucleic acid moleculesof a sample to produce a converted nucleic acid molecules; and ligatinga first nucleic acid adapter comprising a first unique molecularidentifier and a first universal priming site to a first end of theconverted nucleic acid molecules, to produce tagged converted nucleicacid molecules. In certain embodiments, the nucleic acid molecules ofthe sample comprise DNA. In certain embodiments, the nucleic acidmolecules of the sample comprise cell-free DNA. In certain embodiments,the cell-free DNA is derived from a human linear chromosome. In certainembodiments, the human linear chromosome is an autosome. In certainembodiments, the cell-free DNA is derived from blood, serum, plasma,urine, cerebral spinal fluid, or lymph. In certain embodiments,converting unmethylated cytosines to uracils comprises incubation withbisulfite. In certain embodiments, converting the unmethylated cytosinesto uracils in the nucleic acid molecules of the converted nucleic acidmolecules yields tagged converted nucleic acid molecules that are singlestranded. In certain embodiments, the first nucleic acid adapter issingle stranded. In certain embodiments, the first nucleic acid adapteris partially single stranded and partially double stranded. In certainembodiments, the method further comprises performing primer extension onthe tagged converted nucleic acid molecules with a primer that binds tothe universal priming site to produce double stranded, single taggedconverted nucleic acid molecules. In certain embodiments, the methodfurther comprises ligating a second nucleic acid adapter to a second endof the double stranded, single tagged converted nucleic acid molecules,wherein the second adapter comprises a second unique molecularidentifier, to produce double stranded, double tagged converted nucleicacid molecules. In certain embodiments, the sequence of the secondunique molecular identifier is different from the sequence of the firstunique molecular identifier. In certain embodiments, ligating the secondnucleic acid adapter to a second end of the double stranded, singletagged converted nucleic acid molecules creates a gap between a 5′ endof the double stranded, double tagged converted nucleic acid moleculesand a 3′ end of the adapter most proximal to the 5′ end of the doublestranded, double tagged converted nucleic acid molecules. In certainembodiments, the method further comprises amplifying the doublestranded, double tagged converted nucleic acid molecules. In certainembodiments, the amplifying comprises polymerase chain reaction. Incertain embodiments, amplifying the double stranded, double taggedconverted nucleic acid preparation results in the addition of asequencing adapter to the first end, the second end, or both ends of theplurality of nucleic acid molecules. In certain embodiments, the methodfurther comprises determining the nucleic acid sequence of the doublestranded, double tagged converted nucleic acid molecules. In certainembodiments, determining the nucleic acid sequence comprises sequencingthe double stranded, double tagged converted nucleic acid preparationusing a next-generation sequencing method. In certain embodiments,determining the next-generation sequencing method comprises sequencingby synthesis, pyrosequencing, or ion semi-conductor sequencing. Incertain embodiments, the double stranded, double tagged convertednucleic acid molecules are sequenced to a depth of at least 10,000×. Incertain embodiments, sequences containing the same unique molecularidentifier are grouped together for analysis. In certain embodiments,the sequence is compared to a reference sequence or a sequence derivedfrom nucleic acid molecules not treated with ion. In certainembodiments, the first nucleic adapter is immobilized on a solidsupport. In certain embodiments, the solid support is a bead. In certainembodiments, the bead is heat labile. In certain embodiments, the beadis compartmentalized in a liquid droplet. In certain embodiments, themethod is for use in diagnosing or screening a human individual for adisease or disorder. In certain embodiments, the disease or disorder isan autoimmune disease. In certain embodiments, the disease or disorderis screening cancer. In certain embodiments, the disease or disorder isorgan damage, organ disease, or organ failure. In certain embodiments,the disease or disorder is transplant rejection.

In certain embodiments, described herein, is a method of determining amethylation profile in an individual comprising: (a) convertingunmethylated cytosines to uracils in nucleic acid molecules to producesingle stranded converted nucleic acid molecules; (b) ligating asingle-stranded first nucleic acid adapter comprising a first commonbarcode sequence to a first end of the converted nucleic acid molecules,to produce tagged converted nucleic acid strands; (c) performing primerextension to form a second DNA strand duplexed with the tagged convertednucleic acid strands; (d) ligating a double stranded second nucleic acidadapter comprising a second common barcode sequence and a primer regionto the duplexed DNA of step (c), to produce tagged duplex DNA molecules;(e) amplifying the second strand of the tagged duplex DNA molecules toform amplified nucleic acid molecules; (f) determining the sequence ofamplified nucleic acid molecules; and (g) identifying methylation sitesby comparing the sequences of the amplified nucleic acid molecules to areference genome. In certain embodiments, the first, the second, or bothnucleic acid adapters further comprise a unique molecular identifier. Incertain embodiments, determining the sequence of the amplified nucleicacid molecules comprises sequencing the amplified nucleic acid moleculesto produce sequence reads, and collapsing the sequence reads based onthe unique molecular identifier to form a consensus sequence. In certainembodiments, ligating the second nucleic acid adapter creates a gapbetween a 5′ end of the tagged converted nucleic acid strands and a 3′end of the adapter most proximal to the 5′ end of the tagged convertednucleic acid strands in the tagged duplex DNA molecules. In certainembodiments, the second nucleic acid adapter comprises a3′-dideoxynucleotide on one strand.

In certain embodiments, described herein is a method of determining amethylation profile in an individual comprising: (a) convertingunmethylated cytosines to uracils in nucleic acid molecules to producesingle stranded converted nucleic acid molecules; (b) adapter tagging afirst end of the converted nucleic acid molecules a first common barcodesequence, to produce tagged converted nucleic acid strands; (c)generating a second DNA strand duplexed with the tagged convertednucleic acid strands; (d) attaching an at least partially doublestranded nucleic acid adapter comprising a second common barcodesequence and a primer region to the duplexed DNA of step (c) to producetagged duplex DNA molecules; (e) amplifying the second strand of thetagged duplex DNA molecules to form amplified nucleic acid molecules;(f) determining the sequence of amplified nucleic acid molecules; and(g) identifying methylation sites by comparing the sequences of theamplified nucleic acid molecules to a reference genome. In certainembodiments, tagging a first end of the converted nucleic acid moleculescomprises ligating a single stranded nucleic acid adapter comprising thefirst common barcode sequence. In certain embodiments, the second DNAstrand comprises primer extension. In certain embodiments, the singlestranded nucleic acid adapter, the double stranded nucleic acid adapter,or both nucleic acid adapters further comprise a unique molecularidentifier. In certain embodiments, determining the sequence of theamplified nucleic acid molecules comprises sequencing the amplifiednucleic acid molecules to produce sequence reads, and collapsing thesequence reads based on the unique molecular identifier to form aconsensus sequence. In certain embodiments, ligating the second nucleicacid adapter creates a gap between a 5′ end of the tagged convertednucleic acid strands and a 3′ end of the adapter most proximal to the 5′end of the tagged converted nucleic acid strands in the tagged duplexDNA molecules. In certain embodiments, the second nucleic acid adaptercomprises a 3′-dideoxynucleotide on one strand. In certain embodiments,converting unmethylated cytosines to uracils comprises incubation withbisulfite ion. In certain embodiments, converting unmethylated cytosinesto uracils comprises incubation with a cytidine deaminase. In certainembodiments, the method further comprises enriching the convertednucleic acid molecules for one or more target molecules.

In certain embodiments, described herein, is a method of screening anindividual for cancer or an increased risk of developing cancercomprising: converting unmethylated cytosines to uracils in nucleic acidmolecules of a nucleic acid containing sample to produce single strandedconverted nucleic acid molecules; ligating a single-stranded firstnucleic acid adapter comprising a first common barcode sequence to afirst end of the converted nucleic acid molecules, to produce taggedconverted nucleic acid strands; performing primer extension to form asecond DNA strand duplexed with the tagged converted nucleic acidstrands; ligating a double stranded second nucleic acid adaptercomprising a second common barcode sequence and a primer region to theduplexed DNA of step (c), to produce tagged duplex DNA molecules;amplifying the second strand of the tagged duplex DNA molecules to formamplified nucleic acid molecules determining the sequence of amplifiednucleic acid molecules; identifying methylation sites by comparing thesequence of the amplified nucleic acid molecules to a reference genometo determine a methylation profile; and comparing the methylationprofile to methylation profiles associated with cancer to identify ifthe individual has cancer or is at risk for developing cancer.

In certain embodiments, described herein, are kits comprising abisulfite reagent; a nucleic acid adapter comprising a sample IDsequence and a universal primer binding site; and a universal primer. Incertain embodiments, the nucleic acid adapter further comprises a uniquemolecular identifier sequence. In certain embodiments, the kit comprisesa plurality of nucleic acid adapters each comprising a different uniquemolecular identifier sequence. In certain embodiments, the kit furthercomprises a nucleic acid adapter comprising a second different sample IDsequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a flow diagram of an example of a method of preparinga cfDNA library for bisulfite conversion sequencing.

FIG. 2 shows graphically the steps of the method of FIG. 1.

FIG. 3 shows a schematic for labeling ssDNA with a UMI sequence.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are methods of preparing dual-indexed nucleic acidlibraries for methylation profiling using bisulfite conversionsequencing (in which unmethylated cytosines of the nucleic acids areconverted to uracils in the converted nucleic acids). In variousembodiments, the methods use a two-step indexing process to tagbisulfite-treated or enzymatically converted DNA with unique molecularidentifiers (UMIs), wherein a first UMI is added to converted DNA usinga single-strand DNA (ssDNA) ligation reaction and a second UMI is addedin a subsequent processing step (e.g., a double-strand ligation step).The UMIs are used to identify individual DNA molecules and reduce orsubstantially eliminate sequencing and/or amplification-inducedartifacts (based on a consensus among reads sharing the same UMI)thereby increasing the accuracy of DNA methylation analysis.

The UMIs of the present disclosure can serve many functions. The UMI canbe used to identify DNA sequences originating from a common source suchas a sample type, tissue, patient, or individual. The UMIs can be usedto discriminate between a sample treated with bisulfite and a samplethat has not been treated with bisulfite. The UMIs can include universalpriming sites that allow amplification of nucleic acids that have beentagged by a UMI. The UMIs can comprise a unique (e.g., random ordegenerate) nucleic acid sequence which can be used to distinguishbetween nucleic acid fragments in a sample. UMIs can be used to reduceamplification bias, which is the asymmetric amplification of differenttargets due to differences in nucleic acid composition (e.g., high GCcontent). The UMIs can be used to discriminate between nucleic acidmutations that arise during amplification, and mutations that wereinduced by bisulfite or enzymatic conversion of unmethylated cytosinesto uracil.

The UMIs can be present in a multi-functional nucleic acid UMI adapter,which adapter can comprise both a sample ID and a universal primingsite; a sample ID, a universal priming site, and a unique nucleic acidsequence (e.g., a random nucleic acid sequence); or a sample ID and aunique nucleic acid sequence. The sample ID portion can be any suitablelength from 4 to 18, from 5 to 18, from 6 to 18, or from 7 to 18nucleotides. The sample ID tags can be of length sufficient to identifyat least 64, at least 256, at least 1024, at least 4096, at least 16,384or more samples. The unique nucleic acid sequence portion of a UMIadapter can be greater than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, or 18 nucleotides. The UMI adapters can comprise a defined set ofunique nucleic acid sequences.

The UMIs of the present disclosure are provided in the form of nucleicacid adapters that allow the UMIs to be appended to a nucleic acidmolecule using a ligation reaction. The ligation reaction can be ablunt-end ligation. The adapters can be single stranded for ligation toa single stranded nucleic acid molecule. The adapters can be doublestranded for ligation to a double stranded nucleic acid molecule. In acertain embodiment a double stranded adapter can have an overhang thatallows for a gap between a 3′ end of the adapter and the 5′ end of thenucleic acid molecule. Alternatively, the double stranded adaptercomprises a dideoxy nucleotide at the 3′ end of the adapter as shown in240 of FIG. 2. In one embodiment, the methods of the invention are usedfor methylation profiling of cell-free DNA (cfDNA) isolated from abiological sample for detection, diagnosis or screening for a disease ordisorder. The biological sample can be any of blood, plasma, serum,cerebral spinal fluid, interstitial fluid from a biopsy, urine, feces,semen, mucus (e.g., oral or vaginal), sweat, or flash-frozen paraffinembedded (FFPE) tissue samples. In certain embodiments, the biologicalsample is blood, plasma, or serum. The cellular source of the cfDNA canbe either nuclear (chromosomal), mitochondrial or both. In certainembodiments, the cellular source of the cfDNA can be from a tissue suchas the liver, kidney, pancreas, colon, stomach, esophagus, skin, lungs,ovaries, breast, uterus, prostate, testicles, peripheral bloodmononuclear cells, lymphocytes, T cell, B cells, or plasma cells.

FIG. 1 illustrates a flow diagram of an example of a method 100 ofpreparing a cfDNA library for bisulfite conversion sequencing usingadapters containing UMIs. Method 100 includes, but is not limited to,the following steps. In a step 110, a blood sample is obtained andcirculating cfDNA is isolated from the plasma fraction. In a step 115, abisulfite conversion reaction is performed on the purified cfDNA. Forexample, sodium bisulfite treatment converts unmethylated cytosine intouracil, which is replaced by thymine after PCR amplification, while theconversion of methylated cytosine (5-methylcytosine) occurs at a muchslower rate, and therefore most methylated cytosines remain unchanged.

The conversion reaction can be performed using a standard protocol, or acommercially available kit. An exemplar protocol involves starting withisolated DNA, and denaturing the DNA with NaOH at a final concentrationof about 0.3. After the DNA is denatured it can be treated with sodiumbisulfite or sodium metabisulfite at final concentration of about 2M (pHbetween about 5 and 6) at 55° C. for 4-16 hours. This step covalentlymodifies unmethylated cytosines with a sulfite. After conversion the DNAis desalted followed by desulfonation by incubating the DNA at alkalinepH and room temperature, resulting in deamination—and conversion touracil. In one example a commercial kit such as the EZ DNAMethylation-Gold, EZ DNA Methylation-Direct or an EZ DNAMethylation-Lightning kit (available from Zymo Research Corp (Irvine,Calif.)) is used for the bisulfite conversion.

Also contemplated by this disclosure is the conversion of methylatedcytosines to uracils by a method not utilizing bisulfite ion. Cytidinedeaminase enzyme catalyzes the irreversible hydrolytic deamination ofcytidine and deoxycytidine to uridine and deoxyuridine respectively. Insome embodiments, the conversion of unmethylated cytosines to uracils isaccomplished via an enzymatic reaction. In some embodiments, nucleicacid molecules are incubated with cytidine deaminase. In someembodiments, the cytidine deaminase includes activation induced cytidinedeaminase (AID) and apolipoprotein B mRNA editing enzymes, catalyticpolypeptide-like (APOBEC). In some embodiments, the APOBEC enzyme isselected from the human APOBEC family consisting of: APOBEC-1 (Apo1),APOBEC-2 (Apo2), AID, APOBEC-3A, -3B, -3C, -3DE, -3F, -3G, -3H andAPOBEC-4 (Apo4). In some embodiments, the enzyme is a variant of APOBEC(US20130244237). In some embodiments, the conversion uses a commerciallyavailable kit. In one example, a kit such as APOBEC-Seq (NEBiolabs;US20130244237) is used.

The converted nucleotides can be enriched for specific targets ofinterest. In certain embodiments, a converted target nucleic acid isenriched by at least 5-fold, 10-fold, 50-fold, or 100-fold compared tothe unenriched target. Enrichment can be employed for 1 or more targets.In certain embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or moretargets can be enriched simultaneously. Enrichment can be employedthrough an amplification reaction or by binding to specific bait nucleicacids that bind specific enrichment targets. Bait nucleic acids can beimmobilized on a solid support such as a column or substantially flatsurface. Bait nucleic acids can also be immobilized on beads or made ofa magnetic material, agarose, sepharose, or some other bulky materialthat allows for recovery by a magnet, centrifugation, or precipitation.

Still referring to FIG. 1, in a step 120, a first UMI adapter is addedto the 3′-OH ends of the bisulfite-converted ssDNA. For example, a firstUMI adapter is added to the 3′-OH end of a bisulfite-converted ssDNAmolecule using a ssDNA ligation reaction. A first UMI adapter includes,for example, a UMI sequence and a universal primer sequence (e.g., anSBS primer sequence). In one example, Swift Biosciences' Adaptase™technology is used in a ssDNA ligation reaction, wherein a 3′ end G-tailis added to the ssDNA template and an “adaptase” is then used to add apartially double-stranded adapter that includes a 3′-end overhang ontothe template molecule. A polishing step is then used to fill in thebases and repair the nick to attach the adapter. In another example, thessDNA ligation reaction uses CircLigase II (Epicentre) to ligate a firstUMI adapter to the 3′-OH end of a bisulfite-converted ssDNA molecule,wherein the 5′-end of the UMI adapter is phosphorylated and thebisulfite-converted ssDNA has been dephosphorylated (i.e., the 3′ endhas a hydroxyl group). In another example, the ssDNA ligation reactionuses Thermostable 5′ AppDNA/RNA ligase (available from New EnglandBioLabs (Ipswich, Mass.)) to ligate a first UMI adapter to the 3′-OH endof a bisulfite-converted ssDNA molecule. In this example, the first UMIadapter is adenylated at the 5′-end and blocked at the 3′-end. In yetanother example, the ssDNA ligation reaction uses a T4 RNA ligase(available from New England BioLabs) to ligate a first UMI adapter tothe 3′-OH end of a bisulfite-converted ssDNA molecule. In a step 125,second strand DNA is synthesized in an extension reaction. For example,an extension primer that includes a primer sequence that iscomplementary to the universal primer sequence in the first UMI adapteris used in a primer extension reaction to form a double strandedcomplex. The extension reaction uses, for example, an enzyme that isable to read through uracil residues in the bisulfite-converted templatestrand. In a step 130, a second UMI adapter is added to thedouble-stranded bisulfite-converted DNA complex. For example, a secondUMI adapter is a double stranded adapter that includes a UMI sequenceand universal primer sequence (e.g., an SBS primer sequence), whereinone strand includes a 5′-phosphate and the other strand includes a3′-dideoxy nucleotide (i.e., the 3′-end is blocked). The second UMIadapter is ligated to the double-stranded bisulfite-converted DNAcomplex using, for example, T4 DNA ligase in a blunt-end ligationreaction.

In a step 135, the double-stranded bisulfite-converted DNA is amplifiedto add sequencing adapters. For example, PCR amplification using aforward primer that includes a P5 sequence and a reverse primer thatincludes a P7 sequence is used to add P5 and P7 sequences to thebisulfite-converted DNA. In a step 140, the bisulfite-converted libraryis sequenced. In a step 145, the sequencing data is analyzed todetermine methylation sites and patterns. In one example, themethylation sites are determined by comparing the sequence data to areference genome. Comparison of sequence information between thereference genome and bisulfite-treated DNA can provide information aboutmethylation patterns (e.g., cell/tissue-specific methylation,differential hypomethylation and/or hypermethylation, allele-specificmethylation, etc.) that can be used, for example, to infer the tissue oforigin of the cfDNA or identify DNA molecules originating from tumorcells.

FIG. 2 shows a graphical representation the steps of method 100 ofFIG. 1. Namely, in step 110 (not shown in FIG. 2), a blood sample isobtained and circulating cell-free DNA is isolated from the plasmafraction. In step 115, a bisulfite conversion reaction is performed on adouble-stranded DNA molecule 210 to form a bisulfite-convertedsingle-stranded DNA molecule 215. Sodium bisulfite treatment converts anunmethylated cytosine in DNA molecule 210 into uracil (indicated bystars). In one example, an EZ DNA Methylation-Gold or an EZ DNAMethylation-Lightning kit (available from Zymo Research Corp) is usedfor bisulfite conversion. In an alternative step 115, the conversion ofunmethylated cytosines to uracils is accomplished via an enzymaticreaction such as via incubation with cytidine deaminase. In step 120, afirst UMI adapter 220 that is adenylated at the 5′-end and blocked atthe 3′-end is ligated to the 3′-OH ends of bisulfite-converted orenzymatically converted ssDNA molecule 215. In this example, the ssDNAligation reaction uses Thermostable 5′ DNA/RNA ligase (not shown) toligate first UMI adapter 220 to the 3′-OH end of bisulfite-convertedssDNA molecule 215. In step 125, second strand DNA is synthesized in anextension reaction. For example, an extension primer 230 a that iscomplementary to the SBS primer region 230 in first UMI adapter 220 isused in a primer extension reaction to form a double stranded complex235. The extension reaction uses, for example, an enzyme that is able toread through uracil residues (indicated by white stars) in thebisulfite-converted template strand converting the uracil to adenine(indicated by gray stars). In step 130, a second UMI adapter 240 isligated to bisulfite-converted DNA complex 235. Second UMI adapter 240is a double-stranded molecule that includes a second UMI region (e.g., aspecific barcode sequence) and an SBS primer region 250, wherein onestrand (the 3′ to 5′strand, i.e., the “bottom” strand) of second UMIadapter 240 includes a 5′-phosphate and the other strand (the 5′ to 3′strand, i.e., the “top” strand) includes a 3′-dideoxy nucleotide (i.e.,the 3′-end is blocked). Second UMI adapter 240 is ligated tobisulfite-converted DNA complex 235 using, for example, T4 DNA ligase ina blunt-end ligation reaction. Because second UMI adapter 240 is blockedat the 3′-end, a gap 255 is created. In step 135, bisulfite-convertedDNA complex 235 is amplified to add sequencing adapters. For example, anamplification reaction is performed using a forward primer 260 and areverse primer 265. Forward primer 260 includes an SBS primer region 250a that is complementary to SBS primer region 250 and a P5 region 270.Reverse primer 265 includes an SBS region 230 a that is complementary toSBS primer region 230 and a P7 region 275. Because of gap 255 inbisulfite-converted DNA complex 235, the “top” strand is not amplifiedduring the PCR amplification step. A library amplicon 280 now includesP5 region 270, SBS primer region 250, second UMI 245, first UMI 225, SBSprimer region 230, and P7 region 275. In step 140 (not shown in FIG. 2),the bisulfite-converted amplicon library is sequenced. In step 145 (notshown in FIG. 2), the sequencing data is analyzed to determinemethylation sites. In one example, the methylation sites are determinedby comparing the sequence data to a reference genome. Comparison ofsequence information between the reference genome and bisulfite-treatedDNA can provide information about methylation patterns (e.g.,cell/tissue-specific methylation, differential hypomethylation and/orhypermethylation, allele-specific methylation, etc.) that can be used,for example, to infer the tissue of origin of the cfDNA or determinewhich molecules originate from tumor cells.

In another example (not shown), the second UMI adapter 240 is aY-adapter, wherein the UMI sequence is included in the single-strandedportion of the Y-adapter. In this example, each strand ofbisulfite-converted DNA complex 235 is labeled with a different UMIsequence and can be subsequently distinguished.

In another embodiment, methylation sites and patterns can be identifiedusing a (+) bisulfite-converted library and a (−) bisulfite libraryprepared in parallel from aliquots of a single sample. The sample can bedivided into one or more aliquots. In certain embodiments, the samplecan be divided into 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more aliquots.These aliquots can also be divided into replicates in certain casesduplicate, triplicate, or quadruplicate. For example, a (+)bisulfite-converted library is prepared using a first aliquot of a cfDNAsample according to method 100 of FIG. 1 using a plurality of first UMIadapters that include a UMI sequence, a universal (i.e., common) firstbarcode sequence, and a first universal primer sequence (e.g., a SBSprimer sequence). The first universal barcode sequence is used toidentify the library as a (+) bisulfite-converted library and the UMIsequences are used to identify individual DNA molecules. A secondaliquot of the cfDNA is denatured a (−) bisulfite library is preparedaccording to steps 120 through 130 of method 100 of FIG. 1 using aplurality of second UMI adapters that include a UMI sequence, a seconduniversal (i.e., common) barcode sequence, and a universal primersequence (e.g., a second SBS primer sequence). The second universalbarcode sequence is used to identify the library as a (−) bisulfitelibrary and the UMI sequences in the second UMI adapter are used toidentify individual DNA molecules. The (+) bisulfite-converted and the(−) bisulfite libraries are pooled and amplified to add sequencingadapters. Comparison of sequencing data from the (+) bisulfite-convertedlibrary and the (−) bisulfite libraries prepared from a single sample inparallel are used to identify methylation sites and patterns.

In another embodiment of the disclosure the methods solve existingproblems plaguing sequencing of bisulfite treated libraries. One of thechallenges with ssDNA library preparation is losing the duplexinformation, which means after melting the dsDNA, the top and bottomstrand fragments will have different UMI sequences. Thus, in a certainembodiment, the reaction is compartmentalized in droplets or other form,such as beads, and each droplet comprises the same UMI-adapter. Thus allfragments can receive the same UMI adapter. This is shown in FIG. 3.Also, the UMI ssDNA library method is applicable to various librarytypes such as methylation, whole-genome sequencing, and targeted deepsequencing. In a certain embodiment, the UMI adapters are bound to beadsthat are contained in droplets. The beads can be made of a heat labilesubstance such that they can be melted to release the adapter ligatednucleic acids, allowing other steps to be performed in the droplets. Thematerial can be labile at a temperature between 60° C. and 95° C.

In certain embodiments, the nucleic acids that have been subjected tobisulfite conversion or enzymatic conversion of unmethylated cytosinesto uracils have their nucleic acid sequences determined. This sequencedetermination is for the purpose of determining one or more cytosinesthat were methylated in the nucleic acids of the biological sample. Incertain embodiments, a methylation profile is determined. A methylationprofile is the methylation status of a plurality of cytosines in thenucleic acids of a sample. For example, a methylation status isdetermined for 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000 or moredistinct cytosines. A methylation profile can comprise the methylationstatus of cytosines associated with: entire genomic regions thatencompass one or more genes, promoters or enhancer elements, genes thatare normally maternally or paternally imprinted, CpG islands, or CpGshores.

Sequence determination can encompass sequencing stretches of contiguousnucleic acids that are 10, 20, 30, 40, 50, 100, 150, or 300 bases inlength or more. Sequence determination can also encompass thedetermination of distinct polymorphic markers such as single-nucleotidepolymorphisms (SNPs), insertion/deletion mutations (indels), variablenumber tandem repeats (VNTRs), microsatellites, or minisatellites. Otherdisease associated genetic changes can be measured as well, such as copynumber variation. The sequence can be determined using any sequencingmethod such as Sanger sequencing. Sequences can also be determined usinga multiplex assay such as array or bead based hybridization, sitespecific or allele specific PCR. These methods allow the determinationof 100; 1000; or 10,000 different markers simultaneously.

In a certain aspect, sequences are determined using a next-generationsequencing technology. The next generation sequencing technology can bepyrosequencing, sequencing by synthesis, or ion semiconductorsequencing. These methods are capable of sequencing at least 250megabases per machine run. Resulting in at least 10 million discretereads of 25 nucleotides in length or more per machine run. Thesequencing reads can be paired-end reads. Combined with the methods ofthis disclosure this allows for sequencing at a sequencing depth of atleast 10,000×, 20,000×, 30,000×, or 40,000× or more. In certainembodiments, the sequencing depth is between 10,000× and 100,000×;20,000× and 80,000×; 30,000× and 70,000×; or 40,000× and 60,000×.

The sequencing data can be analyzed in a number of ways. The addition ofUMI adapters advantageously decreases the probability of error duringmethylation analysis. By using UMI adapters it possible to differentiatebetween differences in base composition that arise from naturalpolymorphism, amplification error, or bisulfite conversion. Sequencesthat have C>T could arise by many mechanisms, for example, this C to Tchange could be a result of a natural polymorphism that was inherited bythe individual; this could be the result of a mutation introduced duringamplification; or this could be an indication that the cytosine wasmethylated in the original starting material. For example, if eachsequence comprises two different UMI sequences one that signifieswhether the sequence arises from a nucleic acid that has been subjectedto bisulfite conversion or not, and one that signifies a parent nucleicacid (prior to amplification), then it is possible to analyze thesequence for changes in base composition that arise from theamplification or from conversion reaction. If the C>T is primarilypresent in sequences of nucleic acids that have been treated withbisulfite, but absent from sequences of nucleic acids not treated bybisulfite, then this substitution is likely a result of the deaminationof an unmethylated cytosine. Sequences can also be aligned to areference genome or reference sample.

Certain tissues possess tissue specific methylation patterns. Thus,methylation analysis can be used to trace cfDNA to a tissue of origin.The tissue could be a tissue or specific cell-type such as the heart,liver, kidney, pancreas, colon, stomach, esophagus, skin, lungs,ovaries, breast, uterus, prostate, testicles, peripheral bloodmononuclear cells, lymphocytes, T cell, B cells, or plasma cells.

The methods of this disclosure are useful for diagnosis or screening oforgan transplant or organ failure. They can be used to screen for heart,lung, kidney, liver, or pancreatic rejection after transplant from adonor. Increasing levels of DNA derived from a particular organ aftertransplant is indicative of organ failure or rejection. These methodscan be performed on samples taken before and after receipt of an organtransplant. In certain embodiments, the methods can be used forsurveillance post transplant. For example, these methods can beperformed on samples taken longitudinally from a single transplantrecipient at defined intervals. These methods are useful regardless ofthe gender, or genetic relationship of the donor to the recipient.

Alternatively, the methods of the disclosure are useful for diagnosis orscreening for cancer. In certain embodiments, the caner is liver cancer,hepatocellular carcinoma, melanoma, pancreatic cancer, lung cancer,kidney cancer, stomach cancer, esophageal cancer, colon cancer, breastcancer, ovarian cancer, cervical cancer, testicular cancer, prostatecancer, lymphoma, B cell lymphoma, diffuse large B-cell lymphoma,follicular lymphoma, mantle cell lymphoma, small lymphocytic lymphoma,splenic marginal zone B-cell lymphoma, extranodal marginal zone B-celllymphoma of mucosa-associated lymphoid tissue, nodal marginal zoneB-cell lymphoma, lymphoplasmacytic lymphoma, primary effusion lymphoma,Burkitt lymphoma/Burkitt cell leukemia, T cell lymphoma, anaplasticlarge cell lymphoma (primary cutaneous type), anaplastic large celllymphoma,(systemic type), peripheral T-cell lymphoma, angioimmunoblasticT-cell lymphoma, adult T-cell lymphoma/leukemia (human T-celllymphotropic virus type I positive), extranodal NK/T-cell lymphoma(nasal type), enteropathy-associated T-cell lymphoma, gamma/deltahepatosplenic T-cell lymphoma, subcutaneous panniculitis-like T-celllymphoma, multiple myeloma, mycosis fungoides. The methods of thisdisclosure are also useful for diagnosing or screening diseasesassociated with organ damage such as organ failure or autoimmunediseases, such as multiple sclerosis, type I diabetes, lupus, orrheumatoid arthritis.

In addition to diagnosis and screening for cancer, these methods areuseful to gauge response to treatment. For example, these methods can beperformed on samples taken at defined intervals longitudinally from asingle patient receiving chemotherapy, radiation therapy, orimmunotherapy. In a certain embodiment these method are useful fordetermining failure of a particular treatment, or relapse aftersuccessful treatment. The methods of this disclosure are useful incombination with other analyses. Methylation analysis can be combinedwith other analysis of cfDNA. For example, a methylation profile couldbe combined with quantitation of a particular cfDNA, such as a SNP orgene promoter to determine an increase relative to a threshold value orrelative to a sample taken at a previous time. Methylation analysis canbe combined with family history, genomic profiling, metabolic profiling,and other clinically useful tests such as biopsy, PET scan, or MRI.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be understood by those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention.

What is claimed is:
 1. A method of determining a methylation profile ofan individual comprising: a) obtaining a biological sample from theindividual; b) converting unmethylated cytosines to uracils in nucleicacid molecules of the biological sample to produce converted nucleicacid molecules; c) ligating a nucleic acid adapter comprising a uniquemolecular identifier to the converted nucleic acids; d) determining thenucleic acid sequence of the converted nucleic acid molecules; and e)comparing the nucleic acid sequence of the converted nucleic acidmolecules to a reference nucleic acid sequence, to determine themethylation profile of the individual.
 2. The method of claim 1, whereinthe biological sample comprises blood, serum, plasma, urine, cerebralspinal fluid, or lymph.
 3. The method of claim 1, further comprisingenriching the converted nucleic acid molecules, wherein the enrichmentincreases the amount of more target molecules compared to non targeted.4. The method of claim 1, wherein converting unmethylated cytosines touracils comprises incubation with bisulfite ion.
 5. The method of claim1, wherein converting unmethylated cytosines to uracils comprisesincubation with a cytidine deaminase.
 6. The method of claim 1, whereinthe nucleic acid molecules are DNA.
 7. The method of claim 6, whereinthe DNA is cell-free DNA (cfDNA).
 8. The method of claim 1, wherein thenucleic acid adapter comprises a universal priming site.
 9. The methodof claim 1, wherein the nucleic acid adapter is partially singlestranded and partially double stranded.
 10. The method of claim 1,wherein ligating the nucleic acid adapter is performed on singlestranded nucleic acids.
 11. The method of claim 1, wherein the nucleicacid adapter is attached to a solid support.
 12. The method of claim 11,wherein the solid support is a bead.
 13. The method of claim 1, furthercomprising performing primer extension of the converted nucleic acidmolecules, unconverted nucleic acid molecules, or both.
 14. The methodof claim 13, further comprising ligating a nucleic acid adapter to asecond end of the converted nucleic acid molecules.
 15. The method ofclaim 14, wherein ligating the nucleic acid adapter to a second end ofthe converted nucleic acid molecules creates a gap between a 5′ end ofthe converted nucleic acid molecules and a 3′ end of the third adaptermost proximal to the 5′ end of the converted nucleic acid molecules. 16.The method of claim 1, further comprising amplifying the convertednucleic acid molecules before determining the nucleic acid sequence. 17.The method of claim 16, wherein the amplifying comprises polymerasechain reaction.
 18. The method of claim 16, wherein the amplifyingresults in the addition of sequencing adapters to the converted nucleicacid molecules, the unconverted nucleic acid molecules, or both.
 19. Themethod of claim 1, wherein determining the nucleic acid sequencecomprises next-generation sequencing.
 20. The method of claim 19,wherein determining the nucleic acid sequence comprises sequencing to adepth of at least 10,000×.
 21. The method of claim 1, wherein themethylation profile is used to screen for or diagnose cancer.
 22. Themethod of claim 1, wherein the methylation profile is used determine atissue or origin of a cell-free DNA.
 23. A method of determining amethylation profile in an individual comprising: a) convertingunmethylated cytosines to uracils in nucleic acid molecules to producesingle stranded converted nucleic acid molecules; b) adapter tagging afirst end of the converted nucleic acid molecules with a first commonbarcode sequence, to produce tagged converted nucleic acid strands; c)generating a second DNA strand duplexed with the tagged convertednucleic acid strands; d) attaching an at least partially double strandednucleic acid adapter comprising a second common barcode sequence and aprimer region to the duplexed DNA of step (c) to produce tagged duplexDNA molecules; e) amplifying the second strand of the tagged duplex DNAmolecules to form amplified nucleic acid molecules; f) determining thesequence of amplified nucleic acid molecules; and g) identifyingmethylation sites by comparing the sequences of the amplified nucleicacid molecules to a reference genome.
 24. The method of claim 23,wherein the tagging a first end of the converted nucleic acid moleculescomprises ligating a single stranded nucleic acid adapter comprising thefirst common barcode sequence.
 25. The method of claim 23, wherein thegenerating the second DNA strand comprises primer extension.
 26. Themethod of claim 24, wherein the single stranded nucleic acid adapter,the double stranded nucleic acid adapter, or both nucleic acid adaptersfurther comprise a unique molecular identifier.
 27. The method of claim23, wherein the determining the sequence of the amplified nucleic acidmolecules comprises sequencing the amplified nucleic acid molecules toproduce sequence reads, and collapsing the sequence reads based on theunique molecular identifier to form a consensus sequence.
 28. The methodof claim 23, wherein ligating the second nucleic acid adapter creates agap between a 5′ end of the tagged converted nucleic acid strands and a3′ end of the adapter most proximal to the 5′ end of the taggedconverted nucleic acid strands in the tagged duplex DNA molecules. 29.The method of claim 23, wherein the second nucleic acid adaptercomprises a 3′-dideoxynucleotide on one strand.
 30. The method of claim23, wherein the converting unmethylated cytosines to uracils comprisesincubation with bisulfite ion.
 31. The method of claim 23, wherein theconverting unmethylated cytosines to uracils comprises incubation with acytidine deaminase.
 32. The method of claim 23, further comprisingenriching the converted nucleic acid molecules for one or more targetmolecules.